5'Hoxd genes play many roles during limb development and may control the effectors of morphogenesis at late stages. How Hoxd genes guide digit morphogenesis and their downstream targets remain enigmatic. Using genetic approaches in mice we find that, in addition to a role in initiating Sonic hedgehog (Shh) expression, 5'Hoxd genes determine the polarity of the primary limb axis early, and regulate digit pattern and morphogenesis at late stages, after digit condensations have already formed, including joint formation and positioning; a major mechanism by which Hoxd genes regulate digit identity. We previously discovered genetic and physical interactions between 5Hoxd and Gli3 that modify Gli3 repressor (Gli3R) function (and hence Shh output), antagonizing Gli3R and potentially converting it to an activator. We find that Gli3-Hox interactions both modulate the polarity of limb axis formation and regulate the pacing of cartilage vs joint formation in digits, which may have relevance for skeletal homeostasis and disease, as well as skeletal birth defects. ....................................................................................................................................................................................................................................................................................................................Role Role of 5'Hoxd genes and Hoxd-Gli3 interaction in determining polarity of primary limb axis formation: In most vertebrates, the primary limb axis runs through the posterior limb with the ulna/digit4 (d4) condensing first. In urodele amphibians such as axolotl, which retain the ability to regenerate limbs as adults, the anterior limb axis is dominant (radius/d2 appear first). Based on altered expression patterns, it has been proposed that the axis shift in Urodeles results from a failure to expand 5'Hoxd gene expression in the late distal limb. We have analyzed limb axis formation in the 5'Hoxd mutant (Hoxd11-13 deleted) and found that the anterior axis forms first as in urodeles. Furthermore, we find that in compound 5'Hoxd;Gli3 mutants, posterior axial dominance is restored. The 5'Hoxd homeobox transcription factors play roles in replication licensing and cell adhesion. Gli3R, expressed anteriorly, also regulates proliferation and condensation, and antagonizes 5'Hoxd function. We are analyzing how changes in the relative timing and rate of proliferation and of cell aggregation/condensation in different zones of the limb bud are altered in these mutants, and if they correlate with anterior vs posterior axial dominance. We propose that the balance between antagonistic 5'Hoxd-Gli3 functions governs the polarity of primary limb axis formation and are investigating the potential relation between altered axis polarity and regenerative capacity. ....................................................................................................................................................................................................................................................................................................................Role of Hoxd genes and Hoxd-Gli3 interaction in cartilage differentiation and joint formation to determine distinct digit identities: Digit identity remains plastic even after the formation of the digit primordial chondrogenic condensations and is regulated by interdigit zones, which are also late sites of 5'Hoxd and Gli3 expression. We found that genetic removal of several Hoxd genes (d11-d13) results in abnormal joint formation, both loss of digit joints and/or abnormal joint position, as well as short, biphalangeal digits. Collaborating with Marian Ros (Univ. Cantabria) we are also examining the role of Hoxa13 in digit formation. Hoxa13 acts upstream of and induces the late phase of Hoxd13 expression and may have a distinct role in regulating the formation of a normal thumb. The canonical Wnt pathway plays an essential role in joint formation and we find that activated beta-catenin restores normal joint formation in the 5'Hoxd mutant digits. But surprisingly, selective activation of stabilized beta-catenin in the interdigital tissues is required for rescue, suggesting that at least some aspects of beta-catenin and 5'Hoxd function in joint formation occur indirectly, via interdigit signaling. Gli3 (the transcriptional effector of Shh and Hoxd protein interactor) also has striking effects on cartilage differentiation and joint formation in digits. During joint formation in digit precursors, Gli3 mutants form abnormal segments with excessive joint formation extending into the cartilage elements. Genetically, the balance between total 5'Hoxd and Gli3 gene dosage regulates the periodic formation of normal joints and the normal 3 bony segments typical of mammalian digits. Our genetic evidence indicates that the Hoxd-Gli3 balance acts indirectly, from interdigital mesenchyme, to modulate Bmp activity and thereby regulate the periodic appearance of digit elements (phalanges) and joints from a digit tip progenitor pool. We are extending our analysis to determine: 1) targets regulated by Gli3-Hoxd interaction and 2) other signaling inputs that regulate the digit tip progenitor pool to determine phalanx number and size. Collaborating with Steve Vokes (UT-Austin) we will examine the role of the chromatin modifier Prmt5, which is selectively expressed in the digit tip progenitors, in maintaining this progenitor pool. Even in mammals, distal digit tips retain a limited capacity for regeneration and understanding the regulation of this distal digit progenitor pool and it's maintenance will provide new insights relevant to skeletal regeneration potential.